There is an important need for screening methods that can detect and distinguish the relative eye discomfort or "sting" potential of cosmetic, personal care, or pharmaceutical formulations early in their developmental stages. The only currently reliable method available is costly and sometimes painful human clinical eye testing, in which test substances are introduced into human eyes and test subjects report perceived discomfort or stinging. While this may be a critical final clinical test for an ocular product, products in development and formulation stages must also be screened in the same human clinical test, as there are no other available screening options. Available ocular irritation testing can indicate that a product or formulation is "non-irritating", which is a good start. However, current ocular irritation tests are not very predictive of eye sting. Laden (1973) has reported that formulations which had low irritancy often still caused stinging. Hence, the stinging potential of a formulation was often unrelated to irritancy. Van Abbe (1973) has also reported ocular irritancy to be a poor predictor of human reported eye stinging, pain, and discomfort. We hypothesize that this disconnect between measurable irritation and sting potential is due to lack of sensitivity in current irritation tests. Current tests use non-sensitive endpoints such as lack of corneal opacity (i.e. rabbit tests and BCOP) and substantial tissue-wide cell death (i.e. EpiOcular"). EpiOcular" (MatTek Corp, Ashland, MA) is an industry standard ocular irritation test with relatively high sensitivity. EpiOcular " tissues are exposed to test substances and then tissue viability is measured by reduction of a metabolic indicator. Extraction and subsequent measurement of the colorimetric indicator from the entire tissue is the endpoint for overall tissue irritation/cytotoxicity effects. Once the EpiOcular tissues reach 100% viability compared to the negative control tissues (no measurable tissue death), the tissues can no longer be indicative of potential sub-irritation cell death that may be an ocular sting indicator. The Porcine Cornea Confocal Assay, PorFocal, developed by MB Research, likely has the amplified sensitivity to potentially predict human eye sting due to measurement of individual cell death per tissue volume by confocal microscopy. The PorFocal uses waste porcine corneas from the meat industry to assay individual corneal cell death with high sensitivity due to a confocal microscopy endpoint. In PorFocal, test substances are placed directly onto living corneal tissue in culture;therefore solubility of the test substance is irrelevant. PorFocal cultured corneas are maintained in a living state for up to 7 days and are dosed daily with the test substance. This multiple-exposure dosing schedule allows for quantification of extremely mild ocular cell death with additive effects over time. These additive effects are then measured by quantification of individual stained dead cells within the corneal tissue by confocal microscopy. Corneal tissue is imaged in an "optical histological" manner where a series of image "slices" are acquired at increasing depths into the corneal tissue. The images can then be digitally reconstructed to exhibit the entire corneal tissue volume imaged (see Research Strategy). Therefore, extremely low amounts of corneal damage can be quantified because the endpoint is actual individual dead cell number per tissue volume. In Aim 1 seven test substances will be tested in both the EpiOcular " and the PorFocal to determine if the PorFocal assay is more sensitive than the EpiOcular ", the most sensitive in vitro ocular irritation assay (industry standard). This will test the hypothesis that sting is not detected in current in vitro assays due to lack of sensitivity. If PorFocal demonstrates higher sensitivity than the industry standard EpiOcular ", then it may have great potential to predict sting. In Aim 2, commercially available products that are known non-stingers or stingers will be tested in both EpiOcular " and PorFocal. The EpiOcular" will be used to establish whether "stingers" would be considered "non-irritants" by industry standards. If PorFocal can resolve stingers from non-irritants, this may allow for prospective culling of stinging product formulations before final human clinical eye sting testing. Upon further characterization of PorFocal, this test could significantly reduce human test subject pain and distress, and cost/time expenditure during product development and formulation. PUBLIC HEALTH RELEVANCE: This project will test the use of a cultured porcine cornea assay as a highly sensitive pre-clinical screen test to predict human eye-sting potential. A human eye sting screening test is needed for testing during product formulation and development phases, thus allowing for further culling of stinging product formulations before final human clinical eye sting testing. This would reduce overall product development cost and human test subject pain and distress while promoting robust product safety for public health.